An oligonucleotide is a macromolecule comprising a sequence of nucleosides, each of which includes a sugar and a base. Each nucleoside is separated from adjacent nucleosides with an internucleoside linkage, which effectively serves to bond the nucleosides together. The sugar is generally a pentose, most commonly a deoxyribose, ribose, or 2'-0-substituted ribose. A number of different bases can be used, the four most common of which are adenine, cytosine, guanine, and thymine (abbreviated as A, C, G, and T, respectively). The internucleoside linkage is most commonly a phosphate, which may be substituted with a variety of substituents at a nonbridging oxygen atom, most commonly by sulphur or an alkyl, ester, or amide group.
Different methods are used for synthesizing oligonucleotides, including phosphoramidite, phosphotriester, and H-phosphonate methods, each of which is generally known in the field of molecular biology. The phosphoramidite method is described here as an exemplary method. To produce a large number of oligonucleotide molecules with this method, a solid support is provided in a reaction vessel and a large number of DMT-protected nucleosides are fixed to the support. A deprotectant, acting through a detritylation mechanism, is added to remove the DMT from nucleoside, and thus to "deprotect" that one hydroxyl. As a result, the last nucleoside in the sequence has one hydroxyl that is ready to receive a next amidite. Nucleoside phosphoramidites (hereafter "amidites"), dissolved in a solvent such as acetonitrile (ACN), are introduced into the vessel. An activator, such as tetrazole, is also introduced into the vessel with the amidites. The phosphorus in the amidites bonds with the oxygen in the hydroxyl, thus providing support-bound nucleotides. After the support-bound nucleotides are formed, excess amidites are flushed from the vessel with ACN.
An oxidizing agent is added to convert the trivalent phosphorous to pentavalent. After the oxidizing agent is flushed, a capping agent is added to block all the unprotected hydroxyls from reacting with amidites introduced at a later stage. ACN is again introduced to flush out the capping agent.
These steps are repeated a number of times to produce growing, oligonucleotide chains from support-bound nucleosides. Each of the chains should have an identical repeating sequence of nucleosides.
This method (and others) for producing oligonucleotides are time consuming and the materials that are used, particularly the amidites, are expensive and require special handling and disposal after being used. In laboratories, oligonucleotides are synthesized on a scale of about one micromole. One group of machines produced under the name OligoPilot (a trademark of the assignee of the present invention) has improved the process to produce as much as 3-4 millimoles of oligonucleotides. It would be desirable to increase the number of oligonucleotides that can be produced at one time, and to do so efficiently.
In larger quantities, however, the production of oligonucleotides raises several concerns. Because of the interest in using synthesized oligonucleotides for human use, the oligonucleotides must have a high degree of homogeneity. Meanwhile, competing concerns affect the efficient use of materials, particularly the amidites and the ACN. While an excess amount of amidites is needed to ensure that as many as possible of the nascent oligonucleotides react with newly introduced amidites, the quantity of amidites introduced into the vessel should not be too excessive and wasteful. It is also desirable to reduce the amount of ACN that is used, while still flushing out, or at least diluting, leftover amidites as much as possible. If the flushing is insufficient, leftover amidites in the vessel or in various conduits leading to the column can produce nonhomogeneous sequences.
The machine known as the OligoPilot II (also a trademark of the assignee of the present invention) uses a flow-through design in which various conduits, pumps, and valves are constantly filled with liquid. Liquid introduced into a vessel (called a "column" in a flow-through device) displaces previously introduced liquid. This flow-through system is distinguished from a "batch" system in which liquids are introduced into a reaction vessel, the introduced liquids are flushed out, and the steps of introducing and flushing liquids is repeated. In such a batch device, the liquids are provided to the vessel by gas pressure and not with pumps. This approach can be used because a batch process has gaps in the flow of fluid.
In the OligoPilot II machine, first and second eight-way valves, each having eight individually selectable inlet ports, have output ports coupled to inlet ports of a first three-port valve of the type in which one and only one of the inlet ports must be kept open. Each of the two eight-way valves has four inlet ports coupled to receive one of four different types of amidites, and four inlet ports coupled to receive ACN (the flushing agent).
The outlet port of the first three-port valve is coupled to a first inlet port of a second three-port valve of the same type as the first. A second inlet port to this second three-port valve is coupled to a third eight-way valve that has various inputs including ACN, oxidizing agents, capping agents, and activator. The outlet port of the second three-way valve is provided to a valve that is coupled to an inlet side of a reagent pump for pumping liquid to the column through valve at the outlet side of the reagent pump. This last valve is also coupled to two pumps that are dedicated to pumping deprotectant and ACN at a higher flow rate than the reagent pump.
Liquids output from the column are provided through the valve at the pump's inlet side, to a monitor for detecting absorption of light to sense displaced trityl groups, and then to a waste valve that receives one input and has a number of separate outputs for waste. By selecting certain ports on the valves on the inlet and outlet sides of the reagent pump, the liquid can be circulated through the column for a desired time.
In the OligoPilot II machine, to introduce a next amidite into the column, one of the eight-way valves is set to receive a next amidite while another of the eight-way valves is set to receive the activator. The eight-way valves receiving the activator and the amidite are pulsed back and forth to introduce quantities of each alternatively.
To regulate the amounts of the liquids that are provided to the column, each of the pumps is initially calibrated. During operation, the pumps are activated a certain period of time to provide the desired quantities of liquid. Periodically, the pumps must be rechecked and recalibrated to avoid problems that can result from drifting in the pump. For the scale of synthesis involved, such flow calibration is sufficiently accurate for controlling the quantities of reagents delivered to the column. For larger scale synthesis, however, there is a need for more sophisticated means for controlling the delivery of the liquids.